JP2019162134A - Spheroid cell culture well article and methods thereof - Google Patents

Abstract

A well product for culturing and analyzing a spheroid cell mass is provided. The system includes a frame having a chamber (410), the chamber including an opaque sidewall surface, a top opening, a gas permeable transparent bottom (460), and optionally a chamber compartment 420 surface and a second bottom (430); And a cell culture product, wherein at least a portion of the transparent bottom includes at least one concave arcuate surface. [Selection diagram] FIG.

Description

Explanation of related applications

  This application was filed on US patent application Ser. No. 14/087906 filed on Nov. 22, 2013 and filed Apr. 30, 2013, the contents of which are incorporated herein by reference in their entirety. It claims the benefit of priority of US Provisional Patent Application No. 61/817539.

  Any publications or patent documents mentioned in this document are incorporated by reference in their entirety.

  The present disclosure relates generally to cell culture well products and methods of making and using the products.

  In embodiments, the present disclosure provides well products for culturing and analyzing, for example, spheroid cell masses. The well product includes at least one chamber, the chamber having an opaque side wall and a transparent, rounded or concave bottom surface, and the transparent bottom is gas permeable.

  In embodiments, the present disclosure provides methods for making well products and methods for using well products for spheroid cell culture or for cell analysis.

  In embodiments, the present disclosure further provides a multiwell plate product having a well or chamber with an opaque wall and a gas permeable transparent round bottom and a method of making the round bottom multiwell plate product. . For example, a transparent round bottom such as an optically transparent bottom window allows convenient microscopic visualization or inspection of the cultured spheroid cell mass.

  In an embodiment, the present disclosure provides a well plate having a base that has a conical or tapered shape such as 45 ° or approximately the same taper angle from the side wall to the apex (ie, the very bottom of the well). A round-bottomed well or multi-well plate with opaque sidewalls and optically transparent windows or bases for optical visualization of spheroid cell cultures such as in a microscope or similar device provide.

Schematic illustration of a method of combining pre-formed or molded components to make an example of the disclosed product FIG. 5 shows an alternative method of making the disclosed product having an opaque sleeve or chamber wall surface and a pre-formed transparent base insert. FIG. 5 shows an alternative method of making the disclosed product having an opaque sleeve or chamber wall surface and a pre-formed transparent base insert. FIG. 5 shows an alternative method of making the disclosed product having an opaque sleeve or chamber wall surface and a pre-formed transparent base insert. Diagram showing another alternative method of making the disclosed product using strain printing Cross section of single well or single chamber in frame of alternative exemplary product 400 4 is a partial top or plan view of the exemplary product embodiment of FIG. A perspective view of an exemplary perfusion plate device 600 embodiment. Sectional view of an exemplary multispheroid well product 700 embodiment. FIG. 7 is a partial top or plan view of an exemplary product aspect of the multispheroid well product 700 of FIG. Sectional view showing injection molding pin combination 900 that contacts and blocks well bottoms having different shapes Sectional view showing injection molding pin combination 900 that contacts and blocks well bottoms having different shapes Cross section of design instead of pin block Cross section of design instead of pin block Image of cell spheroids in alternative well bottom with different shape Image of cell spheroids in alternative well bottom with different shape Image of cell spheroids in alternative well bottom with different shape Image of cell spheroids in alternative well bottom with different shape

  Various embodiments of the present disclosure will be described in detail with reference to the drawings, if any. Reference to various embodiments does not limit the scope of the invention, which is limited only by the scope of the claims appended hereto. Moreover, any examples set forth herein are not limiting and merely set forth some of the many possible embodiments of the claimed invention.

  In embodiments, the disclosed apparatus and the disclosed methods of making and using the apparatus provide one or more advantageous features or aspects including, for example, those described below. The features or aspects recited in any of the claims are generally applicable to all aspects of the invention. Any single or multiple feature or aspect recited in any one claim may be combined or replaced with any other feature or aspect recited in any other claim .

Definitions "Including" or similar terms mean including but not limited to, ie, including but not exclusive.

  For example, the amount, concentration, volume, process temperature, process time, yield, flow rate, pressure, viscosity, and similar values of components in the composition, and ranges or components employed in the description of embodiments of the present disclosure “About”, which modifies the dimensions, and similar values, and ranges thereof, is used, for example, to prepare a material, composition, composition, concentrate, component, article of manufacture, or formulation of use. Through typical measurements and handling procedures that are performed, through inadvertent errors in these procedures, through differences in manufacturing, source, or purity of the starting materials or components used to perform the method, and similar considerations Refers to the quantity variation that can occur through the matter. The term “about” refers to different amounts of a composition or formulation at a particular initial concentration or mixture due to aging and mixing or mixing of the composition or formulation at a particular initial concentration or mixture. It also contains different amounts due to processing.

  “Optional” or “optionally” means that an event, situation, or structure that is described subsequently may or may not occur, and that the description further describes the event , Situation or structure is meant to include when it occurs and when it does not.

In the embodiment, “consisting essentially of” or “consisting of”
In spheroid cell culture products,
A frame with one or more chambers, eg, wells, each chamber comprising:
An opaque sidewall surface, such as a well having an opaque sidewall;
An upper opening for operational access to the chamber;
A gas permeable transparent bottom surface, and
Optionally, in at least one of the chambers, a porous membrane insert;
A spheroid cell culture product, wherein at least a portion of the transparent bottom comprises at least one concave arcuate surface or a plurality of concave arcuate surfaces in a single chamber,
A method for producing the spheroid cell culture product described above,
Forming a product by combining a gas permeable transparent arcuate bottom surface portion and an opaque sidewall surface portion, achieved in any of the methods disclosed herein or any other suitable method;
Optionally inserting a porous membrane liner in at least one chamber;
Including, and
A method of using a product to culture spheroids, comprising:
Filling the disclosed spheroid cell culture product with a culture medium;
Adding spheroid-forming cells to the culture medium as defined herein;
Including methods,
Can be called.

  The products of the present disclosure, methods of making the products, and methods of using the products, in addition to the components or steps recited in the claims, include the compositions, products, devices, or methods of making and uses of the present disclosure Does not substantially affect the basic and new properties of the method, such as specific product composition selected, specific additives or ingredients, specific agents, specific structural materials or parts, specific irradiation conditions Or it may include other components or steps such as temperature conditions, or similar, structure, material, or process variables.

  As used herein, the singular forms means at least one, or one or more, unless otherwise specified.

  Abbreviations well known to those of ordinary skill in the art can be used (eg, “h” or “hrs” for time, “g” or “gm” for grams, “mL” for milliliters, for room temperature "Rt", "nm" for nanometers, and similar abbreviations).

  The specific values and preferred values disclosed for the components, ingredients, additives, dimensions, conditions, and similar points, and the ranges thereof, are for illustrative purposes only, and are Do not exclude defined values or other values within defined ranges. The devices and methods of the present disclosure are described herein, including any obvious or potential intermediate values and ranges, any value or combination of values, specific values, more specific values, and suitable Can contain a value.

  For example, the formation of uniformly sized tumor cell spheroids can be facilitated by culturing the cells in a round-bottom vessel with low adhesion. Researchers at the Institute of Cancer Research in Sutton, UK, use Corning's ultra-low adhesion (ULA) coated 96-well transparent, round bottom, polystyrene microplates to target Tumor spheroids were generated that allowed validation and drug evaluation (see Vinci et al. BMC Biology, 2012, 10:29). Spheroids increased the biological relevance of tumor cell culture and facilitated various functional analyses. Rather than forming a monolayer of cells, the round-bottom well shape and gravity and ultra-low adhesion coat, or coating, allow the cells to collect and self-assemble into a spheroid shape.

  Using 96-well plates with clear, round bottom, and ULA coated wells gave good results in migration and infiltration analysis due to the optical transparency of the structural polymer, but fluorescence or luminescence detection within this plate The anticancer agent used was not very suitable for evaluation because noise was added to the detection signal by the transparent side wall. Tumor cells can also be transferred to an opaque 96-well plate, but this requires pipetting spheroids, which requires great care, can be difficult and can take a lot of time .

In an embodiment, the present disclosure is a spheroid cell culture product, the product comprising:
Including a frame with a chamber, eg, a well,
An opaque sidewall surface;
An upper opening,
A gas permeable transparent bottom surface;
Have
At least a portion of the bottom surface provides a product having at least one concave arcuate surface, ie, a rounded or curved surface.

  The disclosed well plate with a well base having a conical or tapered shape is more useful for injection molding of the opaque sidewalls of the wells, so far for overmolding with flat inserts (ie bases) This is because there is no need to change the existing flat pin that has been used. Injection molding with existing flat pins can also be accomplished with a well bottom shape having a full radius (ie, a hemispherical or non-tapered base). The optional deformable collar feature of the base can enhance the ability of existing flat pins to achieve a block with the well bottom shape.

  Successful use of well plates with well-shaped tapered bottoms or chevron shapes (eg, 45 ° angle from the side walls to the apex) is used to form cell spheroids. The shape of the tapered well base or well bottom is achieved by placing an opaque well wall on a tapered pre-formed insert (base) using existing flat pins that are typically used for overmolding with flat inserts. It is also possible to overmold. An optional deformable collar feature integrated into or attached to the insert (base) can help block resin flow by the injection molding pin. Alternatively, the well bottom shape can be created by thermally forming a polymer film before overmolding, or using a thermal reshaping process after overmolding. Thermal reshaping allows quick switching to create well shapes with multiple alternative molds.

  The disclosed well plate having a tapered or chevron-shaped base (eg, an angle of approximately 45 °) is advantageous in that it can be overmolded with an insert using, for example, the same pin equipment as an overmold with a flat insert. become. This overmolding can be aided by adding a deformed collar that can bend so that the pins can block the polymer flow. Using flat pins for molding saves time and costs to create this well plate product because there is no need to change the mold cavity pins for different well shapes or different number of well molds Will be reduced.

  Thermal reshaping can be quickly switched to produce well shapes for many different well types. In addition, thermal reshaping allows design flexibility to evaluate the various well shapes in each mold and allows the ability to quickly produce prototypes or product samples for customer evaluation or specification To do.

  According to the well base having a taper angle of about 45 °, a collection of cells that have been sunk due to gravity can be centered so as to form a cell spheroid, and the optical apex can be made smaller to enhance the optical imaging.

  Referring to the drawings, FIG. 4 illustrates a chamber 410, a chamber compartment 420 for receiving a suction pipette 440, and optionally, chambers into an upper chamber 410a and a lower chamber 410b, ie, an upper chamber volume and a lower chamber volume. An exemplary product 400 having a porous membrane 480, for example a high throughput shielding membrane insert or liner, is shown in cross-section. The chamber compartment 420 or chamber extension is not physically separated from the space of the main chamber 410, but rather is a spatial extension or expansion of the main chamber. Thus, dotted line 425 represents the non-physical boundary between the main chamber and the chamber compartment. The chamber compartment and the optional porous membrane provide an excellent arrangement that allows the medium to be aspirated from the well chamber without aspirating the spheroid 450 mass. An optional second bottom 430 or chamber compartment 420 with a stop ledge replaces the liquid medium without significantly disturbing the transparent, arcuate rounded bottom surface area of the lower chamber 410b and the spheroids 450 in the volume 460. For this purpose, a space for receiving the pipette tip 440 of the pipette is provided. Stop ledge 430 prevents the pipette tip from entering the lower spheroid chamber.

  FIG. 5 illustrates some aspects of the exemplary product of FIG. In FIG. 5, which includes a plurality of chambers 410 each having a chamber compartment 420 for receiving the suction pipette 440, a partial top or plan view 500 of the exemplary product of FIG. 4 is shown in the well area A <b> 1. ing. Additionally or alternatively, the well area B1 of FIG. 5 includes a chamber 410 with an optional porous membrane 480 or liner for dividing the chamber into an upper chamber and a lower chamber (410a and 410b, respectively, not shown). This porous membrane 480 covers the underlying or hidden spheroid 450. Products with wells containing porous membrane 480, configured as shown in area B1, provide the functionality of nested 96-well HTS Transwell® spheroid plates To do.

  A significant advantage that a well plate that includes gas permeable round bottom wells with opaque sidewalls and transparent windows can provide, for example, one multi-well plate (for example, to perform an assay to evaluate drug compounds) Spheroids can be formed and visualized in this), eliminating the need to move spheroids from one plate to another, avoiding spheroid transfer steps, saving time and allowing for spheroid loss or disruption And the ability of the spheroids to receive an excellent oxygen supply at the bottom of a well made from a polymer with gas permeability properties at a given wall thickness. The increased availability of oxygen in cells in spheroid cultures is particularly useful for cells with high oxygen demand, such as hepatocytes.

  In embodiments, an article comprising a chamber having at least one concave arcuate surface may include a plurality of adjacent concave arcuate surfaces, including, for example, from 1 to about 1,000 concave arcuate surfaces at the bottom of the same chamber. . See, for example, FIGS. 7 and 8.

  In an embodiment, the product may be a single well or multiwell plate configuration, for example, having multiple “spheroid wells” such as a plurality of depressions or depressions at the bottom or base of each well. Multiple spheroids or spheroid wells per chamber would preferably contain, for example, a single or one spheroid per spheroid well.

  In embodiments, the gas permeable transparent well bottom having at least one concave arcuate surface or “cup” can be, for example, a hemispherical surface, a conical surface with a round bottom, and similar surface shapes, or combinations thereof Good. The well and well bottom are interrupted, eventually terminated with a rounded or curved surface where the spheroids are “friendly”, such as indentations, indentations, and similar concave frustoconical relief surfaces, or combinations thereof. Or the lowest position.

  In embodiments, the opaque sidewall surface (ie, the surrounding) may be, for example, a vertical cylinder or shaft, a portion of a vertical cone that decreases in diameter from the top of the chamber to the bottom of the chamber, or a conical transition. A vertical square shaft or a vertical oval shaft with a square or oval at the top of the well transitioning conically and having at least one concave arcuate surface, ie terminating at a rounded or curved bottom Or a combination thereof. Other illustrative geometric examples include a perforated cylinder, a perforated conical cylinder, a cylinder-to-cone shape, and other similar shapes, or combinations thereof.

  In embodiments, the product can further include, for example, a low adhesion or non-adhesion coating on a portion of the chamber, such as at least one concave arcuate surface.

  In embodiments, the product may further include, for example, a chamber compartment, chamber extension region, or auxiliary side chamber for receiving a pipette tip for aspiration, such as a chamber compartment or chamber extension (eg, side pocket). Can be, for example, an integral surface adjacent to and in fluid communication with the chamber. The chamber compartment may have a second bottom that is spaced from the gas permeable transparent bottom. The chamber compartment and the second bottom of the chamber compartment may be positioned at a higher altitude or a relatively higher position, for example, spaced from the gas permeable transparent bottom. The second bottom of the chamber compartment deflects the fluid dispensed from the pipette away from the transparent bottom to prevent the spheroids from collapsing or disturbing.

  In embodiments, the product further comprises a porous membrane, such as a liner or membrane insert, located within a portion of the chamber, located within a portion of the chamber compartment, or located in both the chamber and the chamber compartment portion. Can be included. The porous membrane is from the first cellular material near the transparent bottom in the lower portion of one or both chambers, in the upper portion of the chamber, or in the upper portion of the chamber formed by the porous membrane, or both. Second cell material, such as a different cell type or a different cell state, located in the chamber can be isolated or separated.

  In embodiments, the at least one concave arcuate surface may be, for example, a hemisphere, or may be a horizontal portion of the hemisphere or a portion of a hemisphere, such as a thin cut of the hemisphere, the diameter of which may be, for example, a selected well shape, each Depending on the number of concave arcuate surfaces in the well, the number of wells in the plate, and similar considerations, including about 250 to about 5,000 μm (ie, 0.010 to 0.200 inches) including intermediate values and ranges ) Other concave arcuate surfaces include, for example, parabolic shapes, hyperbolic shapes, chevron shapes, and similar cross-sectional shapes, or combinations thereof.

  In embodiments, the spheroids can be, for example, substantially spherical, and the diameter can be, for example, about 100 to about 500 μm, more preferably about 150 to about 400 μm, including intermediate values and ranges, depending on the type of cells in the spheroids, for example. More preferably from about 150 to about 300 μm, most preferably from about 200 to about 250 μm. The diameter of the spheroids may be, for example, from about 200 to about 400 μm, and these higher diameters are constrained to account for diffusion (for descriptions of spheroids and spheroid containers, see Achilli, TM et al. Expert Opin. Biol. Ther. (2012) 12 (10)).

In an embodiment, the present disclosure provides a perfusion plate device, the device comprising:
At least one cell culture product,
A chamber, for example with a well, comprising a frame,
An opaque sidewall surface;
An upper opening,
A gas permeable transparent bottom surface;
Optionally the chamber compartment surface;
Have
At least one cell culture product, wherein at least a portion of the bottom has at least one concave arcuate surface, ie a rounded or curved surface;
A medium source well in fluid communication with at least one chamber of the cell culture product, which controllably provides a source of fresh medium to the at least one chamber; and
A waste well in fluid communication with at least one chamber of the cell culture product, controllably receiving the waste medium from cellular metabolism in the at least one chamber;
including.

  FIG. 6 illustrates a perspective view of an exemplary perfusion plate device 600 embodiment. In embodiments, the perfusion plate device described above is a porous liner located within a portion of chamber 610, located within a portion of a chamber compartment (not shown), or located in both the chamber and the chamber compartment. 480 may further include a porous liner from a first cellular material, such as spheroid 450, located near the transparent bottom region 460 in the lower portion of the chamber or chamber compartment, from the upper portion of the chamber, or chamber compartment. The second cellular material 660, such as Caco-2 cells, in the upper part of the chamber or in the upper part of both the chamber and the chamber compartment can be isolated or separated.

  In embodiments, the perfusion plate device described above is in a liquid connection member 635 such as, for example, a stock solution (not shown) or a perfusion plug 655, ie, a tube between the source well 630 and the cell culture round bottom well of the device 600. It may further include a liquid permeable material located, or a perfusion plug 655 located in the liquid connection member 645 between the cell culture round bottom well and the waste well 640, or both. The waste well 640 preferably has an arcuate bottom surface that can assist in removing the waste liquid medium (not shown) using, for example, a pipette, vacuum pump, or similar liquid removal equipment.

  A related but separate cell culture product (ie, FloWell ™ plate) is 2012 to Goral et al., Entitled “Automatic continuous perfusion cell culture microplate consumables”. In US Provisional Patent Application No. 61 / 594,039, now assigned and co-pending application owned by the Applicant, filed on Feb. 2, now in International Patent Application No. PCT / US13 / 24030. It is disclosed. This related application refers to, for example, a microplate for culturing cells using automatic continuous perfusion of liquid medium, including, for example, a well frame that defines a plurality of penetrating cavities. obtain. The microplate can further include a planar substrate connected to the well frame. A planar substrate can provide a bottom surface for a plurality of cavities to form a plurality of wells. The plurality of wells can include a first well, a second well fluidly connected to the first well, and a third well fluidly connected to the first well. The first well can be employed for culturing cells in a liquid medium. The second well can be employed to allow the liquid medium to flow out of the first well or to provide a source of liquid medium. The third well may be employed to accept an inflow of liquid medium from the first well or to accept a waste stream of liquid medium. The second well may be fluidly connected to the first well with a first perfusion membrane. The first perfusion membrane can be disposed between the well frame and the planar substrate and can extend from the outlet portion of the second well to the inlet portion of the first well. The third well may be fluidly connected to the first well with a second perfusion membrane. The first and second perfusion membranes may have a number of pores, for example from about 0.2 to about 200 μm. The second perfusion membrane can be disposed between the well frame and the planar substrate and can extend from the outlet portion of the first well to the inlet portion of the third well. When a perfusion initiating amount of liquid medium is introduced into the second well, the liquid medium flows from the second well through the first perfusion membrane to the first well and from the first well through the second perfusion membrane. And flows to the third well (see, for example, FIG. 5 of the document). This related application further refers to methods for producing microplates and culturing cells.

  FIG. 7 illustrates an embodiment of an exemplary multispheroid well product 700 that is similar to the embodiment of FIG. The multispheroid well product 700 includes, for example, one or more chambers 410, each chamber having a chamber compartment 420 region bounded by a line 425 and a stop ledge 430 for receiving a suction pipette (not shown). Unlike FIG. 4, the bottom of each chamber 410 in FIG. 7 may include a plurality of arcuate depressions 465, which may be either transparent or gas permeable, or both, and each of these depressions is a single unit. Of spheroid 450 may be received or cultured. When cells are seeded at low density in the wells of the device, single spheroids can be formed and cultured in adjacent separated cups. The multispheroid well product 700 may include an optional porous membrane 480 or liner insert to divide the chamber into an upper chamber 410a and a lower chamber 410b.

  FIG. 8 illustrates an exemplary product aspect of the multispheroid well product 700 of FIG. In FIG. 8, which includes a plurality of chambers 410 each having a chamber compartment 420 for receiving a suction pipette (not shown), a top view 800 of the exemplary product of FIG. 7 is shown in well area A1. Yes. The bottom of each chamber 410 includes a plurality of arcuate recesses 465 that can receive or culture a plurality of spheroids 450. In addition or in the alternative, FIG. 8 includes a chamber in the well area B1 that includes an optional porous membrane 480 or liner insert for dividing the chamber into an upper chamber and a lower chamber (410a and 410b, respectively, not shown). 410 is shown. The membrane 480 or liner insert conceals and protects the underlying spheroid 450 and spheroid well 465 from being disturbed or disrupted by force or work above the membrane, such as upon addition or removal of liquid media.

  In embodiments, this product having chambers may include, for example, 1 to about 2,000 chambers, 10 to about 1,500 chambers, including intermediate values and ranges, and each chamber may be any other chamber And each chamber has a single concave arcuate surface. In an embodiment, such a multi-well plate product configuration may include, for example, one spheroid per chamber.

  In embodiments, the at least one concave arcuate surface can include, for example, a plurality of adjacent concave arcuate bottom surfaces in the same well. In embodiments, a single well or multiwell plate configuration may have spheroid wells such as a number of depressions or depressions at the bottom or base of each well. In an embodiment, the product can contain multiple spheroids in separate spheroid wells within a single chamber, preferably including, for example, one spheroid per spheroid well.

In an embodiment, the present disclosure is a product,
Including a frame with a chamber, eg, a well,
An opaque sidewall surface;
An upper opening,
A gas permeable transparent bottom surface;
Optionally, a chamber compartment surface having a second bottom that is remote from the transparent bottom surface;
And having
In a method of making a product wherein at least a portion of the transparent bottom comprises at least one concave arcuate surface, i.e., a transparent, rounded or curved surface,
A method is provided comprising the steps of combining a gas permeable transparent arcuate bottom surface portion and an opaque sidewall surface portion to form the product.

  The method of making the product may further include, for example, inserting a porous membrane in at least one chamber.

  There are several methods that can be used to make an opaque round bottom multi-well plate, including an opaque sidewall and a base with a transparent window. The multi-well plate made in Example 1 or 2 has a flat well bottom shape, which can be subsequently thermally reshaped to create an alternative well bottom shape. A multi-well plate having a flat well bottom shape may be positioned within the nest, for example, having a circular outer periphery of approximately the same dimensions as the well type and with the area of each well bottom position removed, Alternatively, this nesting may change to an apex rounded or rounded at the very bottom of the well, eg about 30 to about 30 including intermediate values and ranges, eg 40 to 50 °, and 45 °. It may have a taper angle of 60 °. A platen with an array of pins, for example with matching 45 ° taper angles, from side walls that slope to the apex rounds, eg 180 to 400 degrees Fahrenheit (82 to 204 degrees Celsius), more preferably Fahrenheit Heat to 250 to 400 degrees (121 to 149 ° C.) and hold the heated platen stationary with the pins positioned over each well. The pin platen is actuated so that the pins descend into the wells of the multi-well plate and contact the transparent, flat well bottom surface. As the pin is advanced, the shape of the well bottom material can be varied by time, temperature, and pressure until the reshaped well bottom material matches the shape of the matching nest. The array of pins may be coated with a release agent such as PTFE so that the reshaped well bottom shape does not stick to the heated pins. Alternatively, the pins need not be coated. The nesting may further comprise a vacuum assist in the nesting to help draw the polymer into the well shape. Cooling air may be injected over the heated pins into the top of the well to slightly cool the hot pins and help release the pins from the reshaped well bottom shape polymer. The nest with a shape matching the platen with the pin array can be quickly replaced to fit many multi-well types such as 384 well and 1536 well. Any number of wells can be reshaped at the same time. In some embodiments, the pin platen may be held stationary while the nest holding the insert is actuated to contact the stationary pin platen. In some embodiments, both the pin platen and the nest holding the insert may be actuated to provide relative movement that provides contact and closure between the pin and the well of the insert.

  The v-shaped well base shape having a taper angle of, for example, 45 degrees inclined from the well side wall to the rounded portion at the apex is superior in terms of spheroid formation and optical measurement compared to the hemispherical well bottom shape. According to the v-shaped well base shape, it can also be overmolded into a well base shape with the same flat pin that can be used to overmold the opaque polymer onto a flat insert. The 45 ° taper angle of the well base allows the pin to contact the tapered sidewall of the bottom of each well base. This can be done with a hemispherical shape, but is more difficult. If the pin cannot be blocked by the sidewalls, the opaque polymer can leak into or onto the transparent well bottom. Alternatively, if the pin cannot be blocked by the side wall, the pin may be made larger than the well and the blocking may be realized in a flat region surrounding each well at the top of the insert. This alternative solution is undesirable because it can create a flat ledge in each well where cells can settle. If the cells settle on the ledge, they cannot contribute to the spheroids located at the center of the well bottom, which may greatly change the experimental results.

  In embodiments, the post-molding equipment may include heated pins, such as a single pin, a row of pins, or an array of pins, for example, these pins may be of a microwell plate to be injection molded, such as a single well, Alternatively, it may match the number and alignment of a plurality of wells in a well column or array. The shape of the end of each pin can have, for example, a 45 ° angle or taper from the side wall and a rounded portion at or near the apex.

  In an embodiment, an injection molded plate having a flat well bottom shape may be positioned and secured within the nest for post molding steps, wherein the heated pin or pin array assembly is For example, it is pushed into the flat well bottom of a microplate to be injection molded. The nesting may have a 45 ° angle that coincides with the location of each well to conform to the pin shape and accept the pin. In embodiments, the platen can hold one or more heated pins and the nest holds the matching well bottom shape. The plane of the pin platen and the nesting plane may be aligned in parallel, and both may be perpendicular to a common axis for proper reversible and reproducible pressing.

  The platen, nest, or both can be quickly exchanged to accommodate other multi-well plate types such as 384 well plates and 1536 well plates. The platen supporting the heated pin is pushed into the matching well of the multi-well plate and selected into the transparent base of the well, a selected shape defined by the shape or cross-section of the tip of the pin, for example Hemispherical, conical, v-shaped, w-shaped, wedge-shaped, and similar shapes, or combinations of these shapes can be formed.

In embodiments, the present disclosure provides a method of culturing spheroids, the method comprising, for example:
Filling any of the aforementioned products with a culture medium; and
Adding spheroid-forming cells to the culture medium;
including.

Materials and Methods There are several methods that can be used to make one or more of the disclosed products. In embodiments, the disclosed product may be, for example, a multi-well plate having opaque well sidewalls and an arcuate or concave rounded base or bottom surface, the base or bottom surface being gas permeable. It may be transparent to visible light or transparent to visible light.

  Assigned U.S. Pat. No. 7,674,346, owned by the applicant, joins a polymer perforated plate to a glass or polymer base, and further uses, for example, heat or radiation to form a gas permeable thin film. References are made to methods of making well plates, such as welding to a perforated plate to form the bottom of a microplate (see, for example, column 2, lines 4 to 44).

  Assigned U.S. Pat. No. 5,759,494, owned by the applicant, refers to a method of making well plates with opaque sidewalls, for example by overmolding inserts, to prevent crosstalk. ing. The gas permeable spheroid plate disclosed herein of polymethylpentene (TPX) may be selected as a suitable base or wall material and may be used in the '494 patent and the method of making the present disclosure. .

  In embodiments, the structure of the gas permeable spheroid plate of the present disclosure is as disclosed in, for example, column 4, line 30 to column 5, line 54 of the '494 patent to further reduce or prevent crosstalk. It may further include one or more ridges arranged in one or more grid patterns. In an overmolded plate that does not include a lattice, the outer periphery of the well, i.e., the film, tends to peel off from the molded plate body. The grid appears to keep the film from peeling off the plate body as it is reshaped. The grid also appears to prevent stress marks from appearing on the film, because without the grid, stress marks appear on the plate.

  US Pat. No. 6,811,752 refers to a method of making a device comprising a microchamber and microfluidic flow and a gas permeable liquid impermeable membrane.

  In one method (insert molding), the bottom or base portion of the multi-well plate, having at least one rounded or cup-shaped or recessed recess in the base, uses a transparent polymer. Injection molded or thermoformed. Next, the molded or thermoformed clear multi-well bottom plate is inserted into the vertical press and then the opaque polymer is injected to place the remaining part of the multi-well plate on the clear rounded well bottom. Form. Instead of insert molding, a two-shot molding process is used.

  Referring again to the drawings, FIG. 1 schematically illustrates a method for joining preformed or molded components. The opaque perforated plate portion 110 (ie, without the well bottom) of an injection molded or thermoformed multi-well plate is joined to a transparent, rounded well bottom plate portion 120 that is injection molded or thermoformed. . This coupling to form the product 130 can be done in any suitable manner, such as placing the well bottom plate 120 (or “insert”) in a vertical press and placing an opaque perforated plate portion on the well bottom plate portion. This can be achieved by molding 110. An alternative fabrication method may be, for example, injection molding the entire multiwell plate using a two-shot molding process. These methods form an integral multi-well plate having a transparent, rounded well bottom and opaque side walls. Any bonding method can be used to attach the pre-formed portions 110 and 120, including, for example, adhesive, ultrasonic, thermal, IR, laser welding, and similar methods, or combinations thereof. included. For example, the subsequent optional well coating with ultra-low adhesion materials and the force of gravity can help cells self-assemble into spheroids. The use of an appropriately sized gas permeable material at least in the transparent, rounded or cup-like well bottom portion of the multi-well plate increases the availability of oxygen in cells within the spheroid.

  The following examples serve to more fully illustrate how to use the above disclosure and to further specify the best mode that is intended to carry out various aspects of the disclosure. These examples do not limit the scope of the present disclosure, but rather are presented for purposes of illustration. The examples further illustrate how to prepare the disclosed cell culture well products.

Example 1 Injection Molding or Thermoforming Method FIGS. 2A to 2C show an alternative method for producing an opaque “sleeve”. FIG. 2A shows a well portion 210 of an injection molded or thermoformed multi-well plate (no skirt) with a rounded or cup-shaped well bottom made with a transparent polymer. FIG. 2B shows a second multi-well plate 220 made of an opaque polymer that does not have a well bottom and that includes an injection molded or thermoformed skirt. The opaque plate 220 has a well diameter that is slightly larger than the well diameter of the transparent plate 210. Slide the transparent part into the opaque part and permanently attach the transparent part by several suitable methods including, for example, adhesives, ultrasonic welding, IR welding, thermal welding, laser welding, and similar methods May be. FIG. 2C shows an assembled plate product 230 that is provided with a transparent plate compression fit within an opaque plate. Opaque plate 220 functions as a sleeve structure and accepts a transparent plate structure, and this combination forms a product with an opaque sidewall and a well having a transparent window at the base of an arcuate or rounded well.

  FIG. 3 illustrates another alternative method of making the disclosed product using strain printing. The transparent polymer sheet 310 is selectively printed with an opaque material to form a print sheet 315 that includes a transparent window region or zone 330. This sheet can be selectively thermoformed to produce a cup (ie, distortion) before or after printing so that the transparent zone 330 of the printed sheet 315 is centered on the bottom of the well. In an embodiment, a finished multi-well plate 340 is formed from a strain-printed polymer sheet using two portions, for example, a perforated plate 350 having a transparent bottom and a mold sheet 315. Once thermoformed into the multi-well plate 360, except for the cup-shaped well bottom transparent zone 370 that forms a window for optical observation at or from the bottom of the well. The whole plate looks opaque.

Example 2
In another fabrication method, an opaque multiwell plate without a well bottom may be molded or thermoformed (ie, creating an opaque “perforated plate”) and then the bottom of the multiwell plate Alternatively, the base portion may be injection molded or thermoformed using a transparent polymer. The perforated plate and base elements may then be attached together using any of a variety of assembly processes including, for example, adhesives, ultrasound, IR, heat, laser welding, and similar processes.

Example 3
In another fabrication method, the entire well of a multi-well plate (without a skirt) may be injection molded or thermoformed with a transparent polymer and then another multi-well plate (including a skirt) with an opaque polymer. ) May be injection molded or thermoformed without a well bottom. The diameter of the well of the opaque plate is shaped to be slightly larger than the diameter of the well of the transparent plate. The opaque plate functions as a sleeve that receives the transparent plate, so that with the transparent window at the base of the well, the opaque plate forms an opaque sidewall.

Example 4
In another fabrication method, an opaque coating may be strain printed outside the transparent polymer sheet, leaving a transparent zone in the central area of the well bottom, for example where one or many cups or depressions are thermoformed. Good. Once thermoformed into a multiwell plate, the entire plate appears opaque except for the transparent zone that forms the optical viewing window.

Example 5: Non-adhesive coating The inner well bottom surface, the wall, or both surfaces have a slightly enhanced property to promote cell attachment, such as perfluoropolymers, olefins, or similar polymers, and mixtures thereof. Optionally, it can be made non-adherent to cells by coating with a polymer that does not have any. Or following assembly of an opaque multi-well plate with a clear, rounded or cup-like well bottom, the inner well bottom such as an ultra-low adhesion material, agarose, non-ionic hydrogel, or similar material, etc. Alternatively, it may be coated with a non-binding material capable of suppressing cell adhesion. The coating may also be applied to the transparent well bottom portion of the multiwell plate prior to combining with the opaque portion. For example, a combination of a low adhesion substrate, curvature of the body and base portions of the well, and gravity can induce self-organization of cells into spheroids, and this cell assembly is more like in vivo It is known to maintain the function of differentiated cells that exhibit a response (see Messner et al., Archives of Toxicology, November 11, 2012). Polymers such as perfluoropolymers or poly-4-methylpentene also provide gas permeability at the thickness typically used in the molding process, and this gas permeability can be beneficial for metabolically active cell types. Typical thicknesses and ranges of gas permeable polymers, including intermediate values and ranges, for example, from about 0.001 inch (25.4 μm) to about 0.025 inch (635 μm), 0.0015 inch (38.1 μm). ) To about 0.03 inches (762 μm) (where 1 inch = 25,400 μm, ie 0.000039 inch = 1 μm). Additionally or alternatively, other materials with high gas permeability, such as polydimethylsiloxane polymer, may provide sufficient gas diffusion at a thickness of, for example, up to about 1 inch (2.54 cm).

  FIGS. 9A and 9B show a cross-sectional view of an injection molding pin combination 900 in contact with and blocking a well bottom having different shapes (9A is v-shaped and 9B is hemispherical). FIG. 9A schematically illustrates how a pin 910 that fits inside the well can be blocked 912 with a 45-degree angle v-shaped well bottom shape 915. FIG. 9B schematically illustrates how the pin 910 can face the potential difficulty of achieving a block 912 with a hemispherical well bottom shape 920.

  FIGS. 10A and 10B show cross-sectional views of an alternative design for blocking by a pin. FIGS. 10A and 10B demonstrate the usefulness of the optional deformed collar 1010. FIG. The wall thickness of the deformable collar 1010 can be, for example, about 0.005 to 0.020 inches (127 to 508 μm) and can be determined, for example, by the elastic modulus of the polymer and the geometrical constraints of the multiwell plate. The deformable collar surrounds the well or surrounds it with a bracket and can be distorted under the pressure of the injection molding pin 910, i.e., bend and stretch elastically, and the contour of the transparent base portion can be injected into the injected side wall. Can be shielded, i.e. sealed or sealed, from the opaque resin used. FIG. 10A shows a pin 910 placed on a common axis 1011 with the pin at the center in the well. FIG. 10B shows a pin 1015 where the pin axis 1012 is slightly offset from the original common axis 1011 to the left of the center, for example, as shown. The deformable collar allows the pin to be more appropriately blocked or sealed to prevent the opaque sidewall polymer stream from flowing into the transparent well bottom. The deformation collar is pushed out more to the left and less to the right, as shown by force vector arrows 1016 and 1017, respectively.

Example 6: Post-Molding Method Another method of making the well plate product of the present disclosure is realized by thermal reshaping. In the thermal reshaping method, for example, a commercially available plate with standard black (opaque) sidewalls and a transparent flat plastic bottom is selected. A rounded bottom using a combination of vacuum on the outside and pressure supplied by a high temperature forming tool with full radius that is pushed into the well inside the well on the other side of the plate and then into the bottom of the well A concave arcuate base or a tapered base having a rounded portion is formed at the bottom of the well. The outer bottom region may be preheated, for example with IR radiation, to aid in the formation of a cup or inner concave arcuate bottom round, or a tapered base.

Example 7: Method of Use, Cell Culture FIGS. 11A-11D show images of cell spheroids in different well bottom shapes. These images were captured with an optical microscope at a magnification of 20 (reference scale is equivalent to 1,000 μm).

  Image 11A shows spheroids in an all transparent control 96-well plate having a full hemispherical well bottom shape (ie, the walls of this comparison plate are transparent).

  Image 11B shows a spheroid in a prototype, thermally reshaped 96-well plate having a v-shaped or 45 ° tapered well bottom shape.

  Image 11C shows a spheroid in an injection molded single well having a v-shape or 45 ° well bottom shape.

  Image 11D shows a spheroid in an injection molded single well having a full hemispherical well bottom shape.

HT29 cells are seeded at a concentration of 10,000 cells per well in wells containing comparative wells (11A), v-shaped wells (11B and 11C) and hemispherical wells (11D) of the present invention, and CO ₂ Was cultured at 37 ° C. for 96 hours in an incubator of 5% and 85% humidity. After 96 hours of culture, the images of FIGS.

  Cell seeding procedures generally included the following steps: trypsinization, counting cells, removing the medium from the cells by centrifugation, and resuspending the cells. The seeding density may be, for example, 10k, 20k, or 30k cells per well. The seeding amount may be, for example, 200 microliters per well. Surveillance images may be recorded including intermediate values and ranges, for example 30 minutes, 24 hours, and 96 hours after sowing.

  In the images 11B and 11C, the spheroid is located at the center of the v-shaped well base. In images 11A and 11D, the spheroid is not the center of the hemisphere. The v-shaped base shape appears to facilitate centering the spheroids in the center of the well base, and this centering aids optical visualization.

  In an embodiment, a rounded gas permeable well bottom can be formed using a thermal reshaping process. In existing methods, the plate can be molded with a flat insert of polystyrene, for example, 0.005 inches (127 μm) thick, which thickness is not considered gas permeable. However, during this thermal reshaping process, the thickness at the radius of the insert can be reduced to, for example, about 0.003 inch (76.2 μm) (photos not included), so that the well bottom is It has a gas permeability comparable to that of a HYPER-product film (also 0.003 inch (76.2 μm) thick polystyrene).

  The present disclosure has been described with reference to various specific embodiments and techniques. However, it should be understood that many variations and modifications are possible within the scope of the present disclosure.

110 Opaque perforated plate portion 120 Well bottom plate portion 130, 230, 400 Product 210 Transparent plate 220 Opaque plate 410 Chamber 410a Upper chamber 410b Lower chamber 420 Chamber compartment 430 Second bottom portion 440 Suction pipette 450 Spheroid 460 Transparent bottom region 465 Spheroid Well 480 Porous membrane

Claims (1)

In cell culture products,
A frame having a chamber, the chamber comprising:
An opaque sidewall surface;
An upper opening,
A gas permeable, liquid impervious transparent bottom surface adjacent to the opaque sidewall surface;
Have a frame,
And wherein at least a portion of the bottom surface includes at least one concave arcuate surface.

Images